1. Field of the Invention
This invention relates to the production of glucoamylase. This invention is also concerned with improving the yield during the fermentation process in the production of glucoamylase.
2. Description Of The Prior Art
Glucoamylase is an enzyme capable of converting starch to dextrose. The use of glucoamylase for producing dextrose and dextrose-containing syrups is well known in the art. Processes using glucoamylase generally fall into three categories. These are the acid-liquefaction-enzyme conversion process, the enzyme-liquefaction-enzyme conversion process, and the enzyme solubilization-enzyme conversion process (the granular starch hydrolysis process as disclosed and claimed in U.S. Ser. Nos. 437,101; 437,264; and 437,452, filed Jan. 28, 1974, and Ser. No. 452,154, filed Mar. 18, 1974, now U.S. Pat. Nos. 3,922,197; 3,922,198; 3,922,199; and 3,922,200, respectively, granted Nov. 25, 1975).
In the acid-enzyme process, starch is liquefied and hydrolyzed in an aqueous suspension containing 20 to 40 percent starch and an acid, such as hydrochloric acid. The suspension is then heated to a high temperature, i.e., a temperature between about 70.degree. C. and about 160.degree. C. and at a pH between about 1 and 4.5 to liquefy and partially hydrolyze the starch. The liquefied and partially solubilized starch will generally have a dextrose equivalent value (D.E.) up to about 20 and preferably up to about 15. Typical acid-enzyme processes are disclosed in U.S. Pat. Nos. 2,305,168; 2,531,999; 2,893,921; 3,042,584 and 3,012,944.
In the enzyme-enzyme process, starch is liquefied and hydrolyzed in an aqueous suspension containing 20 to 40 percent starch and a liquefying enzyme such as bacterial alpha-amylase enzyme at a temperature of from about 85.degree. C. to about 105.degree. C. The dextrose equivalent value of the liquefied and partially hydrolyzed starch is generally less than about 20 and preferably less than about 15. A revolutionary process for preparing a partial hydrolyzates suitable for converting starch to dextrose and dextrose-containing syrups comprises liquefying starch in water with a bacterial alpha-amylase enzyme preparation to a dextrose equivalent value of from about 2 to about 15, heat treating the slurry containing the liquefied starch to a temperature greater than about 95.degree. C., and thereafter converting the liquefied starch with a bacterial alpha-amylase enzyme preparation to a D.E. of up to about 20. This revolutionary process is disclosed and claimed in U.S. Ser. No. 107,436, U.S. Pat. No. 3,853,706.
In the enzyme-enzyme granular starch process, a slurry of granular starch is solubilized by the action of bacterial alpha-amylase (preferably a bacterial alpha-amylase enzyme preparation derived from the microorganism Bacillus licheniformis) under conditions such that the starch is not gelatinized or thinned. The solubilized starch may be thereafter converted to dextrose or dextrose-containing syrups by other enzymes such as glucoamylase.
The partially hydrolysed or solubilized starch products prepared by any one of the three processes mentioned above may then be treated with glucoamylase enzyme preparations to convert the starch hydrolysate to dextrose or dextrose-containing syrups.
The enzymatically converted hydrolysates are then subjected to known carbon and ion exchange refining processes to remove color bodies, odoriferous materials, and constituents which contribute to the ash content of the hydrolysates. Such known treatments involve treating the syrup with activated carbon at an acidic pH (i.e., a pH of about 4 to 6, the pH activated carbon is most effective) and thereafter treating the carbon treated syrup with a strong acid cation exchange resin in the hydrogen form and a weak base anion exchange resin in the free base form.
Glucoamylase is known in the art by many names such as glucamylase, glucogenic enzyme, etc.
Glucoamylase is produced by many types of microorganisms. Certain strains of fungi belonging to the Aspergillus genus such as strains known as Aspergillus niger and certain strains of the Rhizopus and Endomyces genus will produce glucoamylase. The above microorganisms also produce enzymes such as alpha-amylase and transglucosidase. The transglucosidase enzymes are capable of producing saccharide polymers which are unfermentable. Thus, the presence of transglucosidase in glucoamylase enzyme preparations is generally considered undesirable.
There are many methods known in the art for preparing glucoamylase. Many methods deal with removing transglucosidase present in glucoamylase enzyme preparations, e.g., the methods disclosed in U.S. Pat. Nos. 2,976,804; 3,042,584; 3,075,886; 3,117,063; and 3,254,003. A significant advance in the art of preparing glucoamylase is disclosed in U.S. Pat. No. 3,012,944 which discloses and claims a process of mutating a microorganism capable of producing glucoamylase. By this method higher yields of glucoamylase and lesser amounts of transglucosidase are produced.
The commercial production of glucoamylase is conducted in a plurality of steps, beginning with a propagation stage initiated by inoculating spores from a slant of a culture into a pre-sterilized nutrient medium usually contained in a shaker flask. The growth is enhanced by aerating the nutrient medium and maintaining the proper pH and temperature therein. The initial stages are referred to as culture development stages. The microorganisms from the last culture development stage (the seed stage) are inoculated into a large scale fermentor to produce commercial quantities of glucoamylase.
The nutrient medium in at least the final stage of development contains as the primary nutrient substances, a carbon source in the form of a carbohydrate and a nitrogen source such as nitrates or proteinaceous materials. As the carbon source, the nutrient medium generally contains ground corn in amounts ranging up to about 10 percent by weight and varying amounts of starch. Prior to inoculating the nutrient medium with the glucoamylase producing microorganism, the nutrient medium is sterilized by heating to a temperature of at least about 120.degree. C. and holding the medium at this temperature for several minutes. Heating the nutrient medium liquefies the ground corn. However, if the solids content of the ground corn is more than 10%, a viscosity build-up occurs such that the agitators in the fermentor are impeded from their normal function. Heating the nutrient medium to the sterilization temperature without the aid of a hydrolytic catalyst such as a liquefying enzyme results in a phenomenon referred to in the art as "steam bumping". Thus, commercial processes for producing glucoamylase employ the use of an alpha-amylase enzyme in the nutrient medium prior to sterilization to assist the liquefaction of the ground corn and to alleviate the problem of "steam bumping".
While the addition of alpha-amylase during the sterilization process aids in the liquefaction of the ground corn, the use of more than 10% by weight of ground corn is generally impractical due to viscosity build-up.